Table of Contents

High-resolution 3D Chemical Imaging In Vivo

High-resolution, in vivo imaging of brain has garnered a lot of interest lately among the imaging optics, neurophysics, and neurophysiology researchers alike.

Motivation

Traditional techniques like confocal fluorescence microscopy use ultraviolet or visible light for excitation which causes high scattering in the living tissue and results in low penetration and poor signal-to-noise ratio.
These problems can be avoided by utilizing advanced nonlinear optical methods such as two-photon microscopy.
The benefits of nonlinear optics based imaging modalities stem from the two facts: (a) the use of near infrared light for excitation, which scatters significantly less in the living tissue, provides for deeper penetration and higher signal-to-noise ratio and (b) two-photon induced luminescence is extremely localized which enables 3D imaging without requiring a pinhole and provides for a much better photon economy.

Solution

While advances in two-photon microscopy technology allow for faster and deeper imaging in vivo, quantitative chemical imaging still remains challenging.
There exists only a few probes that show sensitivity to biologically relevant chemical species such as dissolved oxygen.
An even fewer of them are water-soluble which makes high-resolution quantitative oxygen imaging in vivo difficult and expensive.
In the search of hydrophilic dyes that are sensitive to dissolved oxygen, several techniques have been developed, such as attaching hydrophilic dendrimers and immobilizing the dye in a polymer matrix.

Our work presents another technique to prepare hydrophobic dyes for aqueous media applications.
We achieve this by encapsulating an oxygen-sensitive hydrophobic dye, \rudppcl,\footnote[2]{Tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride.} in a surfactant, poloxamer 407.
The paper describes the preparation method and the photophysical properties of the probes in Sec.~2 and 3 respectively.
3D intravital imaging of a mouse brain in vivo using ruthenium-poloxamer probes with a commercial multiphoton microscopy setup is shown in Sec.~4. The last section (Sec.~5) discusses the future work with the nanoprobes and some potential applications.

Highlights

Signal-to-noise Ratio in Multiphoton Fluorescence Lifetime Imaging

Signal-to-noise ratio (SNR) is important for fluorescence lifetime imaging, especially for multiphoton excitation, which generally has weaker fluorescence intensity than the traditional one-photon confocal excitation.

Motivation

During fluorescence lifetime imaging, very few photons are detected per pixel; therefore the signal quality is usually limited by the photon quantum noise. This problem is more severe for the multiphoton excitation fluorescence, since multiphoton processes result in lower fluorescence intensity than single-photon excitation. This research is aiming at finding ways to improve the SNR in the lifetime acquisition method.

Solution

To quantify and evaluate the SNR performance of the multiphoton fluorescence lifetime imaging method, two figure-of-merits are used in this research: the photon economy (F-value), and the normalized SNR. F-value quantifies the sensitivity of the lifetime acquisition approach. It is always larger than 1, and the closer the value to 1, the better the SNR performance. While the normalized SNR is positvely correlated with the performance. SNR depends on both the waveform of the modulated light on the detector side, and the lock-in technique used on the detector side. Theoretical analysis, mainly the error propagation method, and Monte-Carlo simulations are conducted to quantify the figure-of-merits for a variety of modulation waveforms (source side) and lock-in techniques (detector side) respectively.